ProjectSummary Neuroblastoma (NB) is an often fatal form of childhood cancer caused by aberrant development of the sympathetic nervous system (1). The majority of patients have widely disseminated disease at diagnosis, and despiteintensemulti-modaltherapy,thesurvivalrateforhigh-riskNBremainslessthan50%,andrelapsedNB isalmostuniversallyincurable.ThereisanurgentandunmetneedtoimprovetheoutcomesofchildrenwithNB, which constitutes our long-term goal. Recently, antibody-based immunotherapeutic targeting of GD2 in NB showedsignificantsurvivaladvantagesinaphaseIIItrial,andisnowbeingadministeredtohelppreventdisease recurrence(2).However,identificationofidealanticanceragentsthatspecificallytargettumorcellsandminimize adverseeffectsresultingfrominjurytohealthycellsremainsasignificantchallengeinsolidtumors,includingNB whereGD2alsoexpressedonpainfibers.Ourobjectivehereistoidentifyanddelivernewimmunotherapeutic targets for high-risk and/or relapsed NB. Our central hypothesis is that an integrative proteogenomic strategy utilizing highly annotated patient-derived NB samples coupled with rigorous experimental validation will define clinicallyactionableimmunotherapeutictargetswithalargetherapeuticindex.Ourinitialstudieshaveidentified DLL3,CNTN1,GFRA2,UNC5CandADAM22asproteinspreferentiallyexpressedonthecellsurfaceofNB.We will build on these data, and test our central hypothesis in specific aims: (1) Define the cell surface protein landscapeofhigh-riskandrelapsedNBusingDNA/RNA-sequencingandmassspectrometry(MS).Specifically, wewillprofile10NBpatient-derivedcelllinesand20NBtumors(10patientderivedxenografts,5primaryand5 relapse patient tumors) by MS. Computational integration of proteomic and transcriptomic with UniProt annotations have allowed us to filter lowly expressed proteins and those lacking an extracellular domain. In addition,RNA-sequencingdata(2,242NBprimaryandrelapsedtumorsand7,859normaltissues)allowsusto ensureno,orminimal,expressioninnormaltissuesandprioritizecandidatetargetsforfurtherstudy.(2)Validate and assess biological relevance of candidate immunotherapeutic targets. Here, we will verify cell surface expressionofcandidatetargets(seepreliminarydata)usingacombinationofwesternblot,immunofluorescence (IF), flow cytometry and immunohistochemistry (IHC). We will validate cell surface expression in a panel of normaltissuesandNBsamplesbystainingtissuemicroarrays(TMA)incollaborationwiththeCHOPPathology Core. To determine the biological relevance, we will perform in vitro studies following genetic manipulation of candidategenesincludingCNTN1.Thisworkwillhaveasustainedandlastingimpactonthefieldbyproviding thefirstcharacterizationofcellsurfaceproteomeofdiagnostic/relapsedNB.Weexpecttoidentifyatleastone noveltargetthatwillsubsequentlyundergoantibody-basedand/orchimericantigenreceptor(CAR)Tcell-based development.Themethodswedevelopcanbeappliedtoothermalignanciesimpactingmanycancerpatients.